Methods which have been developed for detecting infectious organisms include traditional methods of identifying the physical and chemical characteristics of pathogens by cultivation, and methods of detecting the specific genetic characteristics of pathogens. The methods for detecting genetic characteristics include restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis, arbitrarily-primed polymerase chain reaction (AP-PCR), repetitive sequence-based PCR, ribotyping, and comparative nucleic acid sequencing. These methods are generally too slow, expensive, irreproducible, and technically demanding to be used in most diagnostic settings. All of the above-mentioned methods generally require that a cumbersome gel electrophoretic step be used, that the pathogen be grown in culture, that its genomic DNA be purified, and that the sample not contain more than one type of organism. These limitations also apply to recently developed detection methods which employ high density microarrays (Salazar et al., Nucleic Acids Res. 24:5056-5057, 1996; Troesch et al., J. Clin. Microbiol. 37:49-55, 1999; Lashkari et al., Proc. Natil. Acad. Sci. U.S.A. 94: 13057-13062, 1997). Meanwhile, pyrosequencing is a method of DNA sequencing based on the “sequencing by DNA synthesis” principle, which relies on the detection of pyrophosphate release on nucleotide incorporation, unlike the traditional Sanger sequencing method. In the pyrosequencing method, four deoxynucleotide triphosphates (dNTPs) are sequentially added one by one during polymerization. PPi attached to the dNTPs being polymerized emit light by enzymatic reactions, and the emitted light shows a signal peak according to the reaction order of each of the sequentially added dNTPs, in which the peak shows a pattern which is high or low in proportion to the number of the reacted dNTPs, such that the nucleotide sequence of the pathogen can be determined. In recent years, methods of detecting pathogenic bacteria or viruses in clinical samples based on pyrograms obtained by pyrosequencing of the PCR products of sequences specific to the pathogens have been used (Travasso, C M et al, J. Biosci., 33:73-80, 2008; Gharizadeh, B et al., Molecular and Cellular Probes, 20, 230-238, 2006; Hoffmann, C et al., Nucleic Acid Research, 1-8, 2007).
In the pyrosequencing technique, however, nucleotide sequencing is performed according to the dispensation order of dNTPs, and a nucleotide in a template, which is absent in the dispensation order, does not react, and thus does not form a peak. However, when identical nucleotides in the dispensation order continuously appear, the heights of the peaks are determined according to the intensities of light emitted. Accordingly, when various pathogens exist in the same sample, the peaks of the nucleotides of the various pathogens appear overlapped, thus making it difficult to identify the genotypes through the interpretation of pyrograms. Particularly, as the number of repetitive sequences increases, the peaks of the anterior sequences become relatively lower. Thus, in the case of infection with multiple pathogens, it is difficult to detect a peak according to the degree of infection with each pathogen.
Accordingly, the present inventors have made extensive efforts to enable the genotypes of interest to be identified by unique and simple pyrograms obtained when performing genotyping using pyrosequencing. As a result, the present inventors have found that, when an ID sequence, which has an ID mark, a signpost and an endmark while existing independently of the specific sequence to be typed, is linked with the specific sequence and is used to perform pyrosequencing, a unique and simple pyrogram can be obtained for each genotype, thereby completing the present invention.